This invention relates to proteolytic enzymes and to a method of producing such enzymes by culturing T. reesei MCG80 under suitable conditions. These enzymes have been found suitable for tenderizing animal muscle used for food purposes.
The increasing cost of animal meats for human consumption has heightened interest in the development of meat tenderizers which can improve the acceptability of lower grade meats. Protein chemistry is playing an increasingly important role in the development of methods for the selective tenderization of muscle used as food (meat, fish). At present, commercial meat tenderizers contain the plant protease papain which is relatively indiscriminate in its attack on muscle proteins and leads to extensive degradation of the structural proteins, myosin and actin as well as the other proteins associated with myofibrils.
Research into the post-mortem aging of bovine muscle has shown that there are two important phases which affect texture. The first, which is accompanied by a drop in adenosine triphosphate (ATP) concentration and a fall in pH from a physiological value of about 7.1 to about 5.3, results in rigor. This phase is complete in about two days with no significant change in muscle fiber surface ultrastructure occurring except shortening. The second stage of aging, the resolution of rigor, is noticeable in about five days. Transverse breaks appear in muscle fibers and elongation of sarcomere lengths occur with a decrease in tensile strength of fibers. At the myofibrillar level, Z--line degradation occurs and the interactions between actin and myosin weaken. This second stage of aging results in tenderization and has recently been shown to be due, in a large measure, to selective proteolysis of the muscle proteins by cathepsins, the endogenous proteolytic enzymes of muscle.
Cathepsin D, the major acidic proteolytic enzyme of muscle, has been shown to produce structural change in muscle under post-mortem conditions which were similar to those observed during aging. Treatment of bovine muscle with bovine spleen extract, a rich source of Cathepsin D, produced structural changes similar to those occurring during natural aging. The texture of the muscle treated with spleen extract was similar to that of naturally aged and tenderized muscle.
The presence of large amounts of connective tissue adversely affects the texture of meat. Since muscle has very little endogenous proteolytic activity against connective tissue, aging does little to improve the texture of muscle containing large amounts of connective tissue. Meat tenderizers containing papain possess significant activity against connective tissue or proteins which could be beneficial in modifying the texture of meat at high temperatures (55.degree.-65.degree.) but the indiscriminate proteolytic action of this enzyme on the myofibrillar proteins leads to overall inferior textural properties. By contrast, the spleen extract also has considerable activity against connective tissues but is quite selective in its action on the myofibrillar proteins. This recommends it both as a superior tenderizer for muscle and for upgrading inferior cuts of meat.
A disadvantage to the use of catheptic enzymes obtained from spleen is the difficulty of obtaining the enzyme at a reasonable cost in a reasonable yield. The production of similar enzymes from microbial sources could be an attractive solution to this problem by producing large quantities at low cost.
Interest has grown in the isolation and utilization of extracellular proteolytic enzymes from microbial sources as reagents in laboratory and clinical procedures and for industrial processing. These enzymes are of several types, including, serine proteases, metalloproteases and acid proteases.
This application describes the preparation, isolation and characterization of an aspartic acid protease from the fungus Trichoderma reesei with properties similar to the animal protease Cathepsin D. It also describes the use of the protease as a meat tenderizer and its use to increase the water absorbing capacity of stored freeze-dried meats.